Currently a number of studies emphasize the importance of developing culturing systems that mimic similar organization of cells similar to that appearing in intact tissue (Cukierman E et al., 2001; Griffith and Naughton, 2002; Schmeichel and Bissell, 2003). Such a culturing system is believed to create a more appropriate dynamic system for research and clinical approaches. An environment that encourages cell growth into a three dimensional (3D) structure (cell aggregates) without the requirement of other manipulation or serial culturing is not possible with standard culture plates. Cells cultured in such a medium might also induce changes in cell geometry necessary to favor proliferation responses. The maintenance of stem cells properties in culture and the generation of larger numbers of stem cells are essential criteria for their clinical application.
Hydrogels such as agarose, alginate, gelatin, fibrin, and others have been used in 3D osteoblast cell culturing, and in the in vivo delivery of such cells to implantation sites (Trojani C et al., 2005). These studies demonstrated good cell viability and opportunity for inducing cell differentiation in the hydrogel cell culture. However, mechanical damage to cells, possible hindrance of cell migration and, consequently, low integration percentage in the implantation site have to be taken into account when using gel-encapsulated cells for in vivo cell delivery applications.
It is evident from several studies that complex cell to cell and cell to extracellular matrix interactions are taking place in living tissue (Yamada and Clark, 2002; Gumbiner B M, 1996; Ohlstein B et al., 2004). Engler (2006) found that matrix stiffness can modify the direction of differentiation of human mesenchymal stem cells. They showed that soft substrates mimic the elasticity of brain tissue, whereas stiff substrates mimics the matrix of bone in governing the differentiation of stem cell. A major problem is to identify substrates that mirror the original three-dimensional cell organization in a given living tissue to allow the development an efficient in-vitro 3D cell culture system.
Hydroxypropyl-methyl cellulose (HPMC) has been employed in combination with biphasic calcium phosphates (BCP) to produce injectable bone substitute for cavity filling in bone repair (Grimandi G et al., 1998; Lerouxel E et al., 2006). Grimandi et al. demonstrated that a combination of HPMC and calcium phosphates is not toxic in vitro, and they observed inhibition of cell growth as they used the pre-incubated media extraction on HPMC and calcium phosphates to evaluate cell growth. Lerouxel (2006) used a injectable calcium phosphate scaffold (ICPS, consisting of a mixture of BCP calcium granules and a cellulosic polymer derivative) as a carrier for bone marrow graft to fill defect areas in an animal model for irradiated bone defect. They showed a significant increase in bone repair when using a mixture of bone marrow cells and ICPS to fill bone defects compared with grafting ICPS only.
Bone tissue regeneration in dental, plastic/reconstructive and orthopaedic applications aims at coping with different kinds of injuries by which bone tissue has been impaired. A graft material is placed in or on a graft site followed by covering the graft material and the site with a barrier. New bone growth is induced at the site by host resorption of the graft material. The graft material must be able to induce bone formation in the site. The barrier material should be biodegradable. Titanium and tantalum are biocompatible metals used as bone substitute and/or in bone replacement and/or for fixtures in implant and/or prosthetic surgery.
Dental implants fixed in a jaw are frequently used for teeth replacement after parodontitis or other causes of loss of teeth. For a successful fixation of a dental implant the jaw must contain a sufficient amount of bone material. Amelogenin (Emdogain®) is a matrix protein mixture used in the regeneration of lost attachment of teeth. The agent precipitates on the tooth root surface and induces stem cell colonization resulting in root attachment. Osteoporosis is a result of an imbalance between bone resorption and bone formation, in which osteclasts resorb bone tissue faster than it is formed by osteoblasts. Osteoporotic bones are more prone to fracture. Typical osteoporotic fractures occur in the vertebral column, the hip and the wrist.
A silanized hydroxypropyl methyl cellulose (Si-HPMC) has been used for 3D cell culture of human chondrocytes and human osteogenic cells. (Vinatier C et al., 2005; Vinatier C et al., 2007; Trojani C et al., 2005). The Si-HPMC scaffold is however not water-soluble and thus cannot be removed by rinsing the cultured osteogenic cells with aqueous media such as body fluids.
WO 98/041617 discloses an aqueous solution of hydroxypropyl-methyl cellulose (HPMC) for use as a medium to stimulate the growth of cultured pancreatic beta-cells. EP 1 303 586 A1 discloses an aqueous solution of HPMC as a DNA/RNA-embedding medium.
Collagen type I is the most abundant bone matrix protein. It constitutes ninety percent of the total organic matrix of mature bone cells, which is responsible for the strength of the tissue. Osteocalcin is a later marker for osteogenesis and is involved in matrix mineralization and its expression closely mirrors the course of mineralization. It has a regulatory role in balancing bone mineralization (Termine J D et al., 1981). CD44 is considered as a marker for osteocytes. Expression of the CD44 gene has been detected in all stages of osteoblastic cell differentiation. It has been suggested that CD 44 gene expression it might play a role in osteogenesis and in bone tissue organization (Jamal and Aubin, 1996).
JP 7079772 A discloses a cell culture liquid comprising a water soluble polymer such as methyl cellulose or carboxymethyl cellulose for use in the production of cell spheroids from human fibroblasts of a controlled size. The spheroids so produced are intended for in-vitro assessment of toxicity of drugs.
JP 2003304866 A1 discloses float-cultured cell aggregates of chondrocytes and normal bone cells in a container with non-adhesive walls.
Mesenchymal stem cells (multipotent stromal cells) are multipotent cells capable of differentiating into a variety of cell types, such as osteoblasts and chondrocytes. A problem in the culture of mesenchymal stem cells and osteoblasts are the low expansion levels obtained with cell culturing methods known in the art.